Development of the endocrine system in the gut is poorly understood. Enteroendocrine cells are divided into numerous subtypes; each defined by the hormone product they secrete and their secretory granule morphology. In this project we will analyze the cholecystokinin (CCK)-expressing cell lineage. CCK is an abundant marker in the proximal intestine that exhibits strict spatial and cell-type specific expression. The approach taken by this grant is to define gene regions capable of directing CCK expression in vivo in transgenic mice, and to test those gene segments in vitro in cell culture model systems. The goal is to identify the transcription factors that bind to the DNA sequences that regulate endocrine-cell specific gene expression. The hypothesis is that transcriptional regulators of CCK will be key participants in endocrine cell differentiation in the small intestine. Previous studies have identified a 6.5-kb segment of the CCK gene that can direct appropriate expression, and a 110-kb Bacterial Artificial Chromosome clone that directs consistent gene expression in the intestine. Specific Aim 1 will focus on further refinement of the CCK DNA sequences that contain important regulatory information by gene transfer into transgenic, mice, and by transient transfection into the CCK-expressing enteroendocrine cell line STC-1. One candidate regulator of CCK is the basic helix-loop-helix transcription factor NeuroD/BETA2, as suggested by the observation that NeuroD 1/BETA2-deficient mice do not express intestinal CCK. In Specific Aim 2 we will test the hypothesis that this transcription factor activates CCK gene expression by overexpressing NeuroD1/BETA2 in immature enterocytes in transgenic mice, and by cotransfection of NeuroD1/BETA2 and CCK constructs in HeLa cells. Early expression during intestinal organogenesis suggests that CCK is a marker of endocrine precursor cells. Moreover, there is evidence that CCK and secretin, another endocrine product of the proximal intestine, share a common endocrine cell lineage. In Aim 3 we will test whether secretin-cells are derived from CCK-expressing precursor cells using a novel cell lineage marking experiment with a CCK-Cre knock-in mouse and the ROSA26 indicator mouse line. Currently, very little is known about the mechanisms of commitment and differentiation of intestinal stem cells to endocrine cell lineages. Since differentiation can be disrupted in pathologic conditions such as intestinal metaplasia, an understanding of the molecular signals that regulate CCK will contribute to our understanding of aberrant cell differentiation in the gastrointestinal tract.